1. Field of the Invention
The present invention relates to methods of delivering nucleic acids into cells in vivo or in vitro using a nucleic acid binding molecule containing a multimeric and/or spacer-incorporated protein transduction domain (PTD). The invention also relates to novel compositions that contain a nucleic acid complexed or conjugated with a nucleic acid binding molecule. The invention further relates to a method of inhibiting expression of a target gene, as well as determining the function of a target gene.
2. Background Art
RNA interference (RNAi) refers to a process in which short RNA fragments interfere with messenger RNA (mRNA), an important mediator of gene expression, by inducing the degradation of mRNA, to block the synthesis of proteins as mRNA products. When the short RNA fragments base pair with an mRNA, a double-stranded RNA is formed that is degraded in cells. The selective effect of RNAi on gene expression makes it a valuable research tool when investigating the function of a specific gene. Also, RNAi has been frequently used in the development of new drugs which specifically suppress the expression of target genes.
Dependent on origin, short RNA fragments are classified as small interfering RNA (siRNA) when they are derived from exogenous sources (Elbashir, S. M., et al., Nature 411:494-498 (2001)), and microRNA (miRNA) when they are produced from RNA-coding genes in the cell's own genome.
The use of siRNA can be largely divided into two categories: (1) siRNA or short hairpin RNA (shRNA), produced in vitro by chemical synthesis or biological synthesis, is delivered directly into cells; and (2) various DNA vectors capable of expressing siRNA are injected into cells, whereby the cells produce siRNA (see, e.g., U.S. Pat. No. 6,278,039; U.S. Application No. 2002/0006664; WO 99/32619; WO 01/29058; WO 01/68836; and WO 01/96584). Various application techniques based on these two categories can be used such that their advantages are effectively utilized. For the latter one, recent attempts have also been made to deliver DNA vectors, which can make siRNA, to down-regulate genes which are turned on in diseases such as cancer (Meyer, M. and Wagner, E., Hum. Gene Ther. 17(11):1062-1076 (2006)).
In RNAi, the efficiency of delivering nucleic acids into cells in vivo or in vitro determines the efficiency of RNAi. However, the efficiency of delivering siRNA or DNA vectors for siRNA into cells remains a major impediment for the practical application of the two techniques. This is because nucleic acids such as RNA or DNA are too large to permeate the cell membrane. In experiments, these nucleic acids can be introduced into cells without the need for physical or chemical means, even though the mechanism thereof is not clearly found. However, the delivery of nucleic acids is difficult to apply in practice because it has excessively low efficiency.
One method, which is most frequently used in in vitro experiments in laboratories, is to use liposome to aid delivery of nucleic acids. In the 1980s, cationic liposome was developed, which has greatly improved transfection efficiency compared to neutral liposome. However, this efficiency is high in in vitro experiments and is greatly decreased in vivo due to blood or body fluids. Also, liposome itself has strong toxicity, which makes it difficult to apply liposome in large amounts, thus limiting the application thereof in the human body.
Another method for delivering nucleic acids into cells uses viral vectors. The viral vector can be used as an effective carrier, but was found to have serious side-effects, such as cell carcinogenesis, in addition to the ethical problem of introducing foreign genes into human cells, and thus extensive clinic studies are strongly required to ensure safety. For this reason, in current circumstances, the in vivo introduction of viral vectors cannot be reliably used clinically or in industry despite many studies.
Other methods of physically delivering naked nucleic acids (i.e., nucleic acids without any other components to aid their delivery) directly in vivo, for example, by electroporation and hydrodynamic injection, have also been studied. The results of recent studies showed that the delivery of naked RNA into veins, abdominal cavities or eyeballs can knock down the expression of specific genes (Herweijer, H. and Wolff, J. A., Gene Ther. 10(6):453-458 (2003); Hagstrom, J. E. et al., Mol. Ther. 10(2):386-398 (2004)). However, the characteristics of these methods limit their practical applications.
In addition, studies focused on the use of nanoparticles including polyethylenimine (PEI) or the like, as delivery vectors, have been actively conducted.
Other than low efficiency in delivery, another impediment to using siRNA in practice is that it has a short in vivo half-life, which requires the use of increasing amounts thereof. Attempts have been made to modify the phosphate backbone of RNA into phosphorothioate or the like such that it has resistance to RNAse. Moreover, studies focused on increasing the half-life of RNA by modifying RNA with polyethyleneglycol (PEG), cholesterol or the like have also been conducted. However, despite such various attempts and studies, low nucleic acid delivery efficiency remains a major problem in the practical use of RNAi.
Recently, the use of a protein transduction domain (PTD) has been proposed. PTDs are low molecular-weight peptides that are useful for the delivery of biologically active molecules into cells (Viehl C. T., et al., Ann. Surg. Oncol. 12:517-525 (2005); Noguchi H., et al., Nat. Med. 10:305-309 (2004); and Fu A. L., et al., Neurosci. Lett. 368:258-62 (2004)). Various PTDs are known, but in most cases, the number of positively charged amino acids in a PTD is very high. The most commonly known PTD is Tat of HIV, and in addition, there are Antp, VP22, synthetic polyarginine and the like. Recently, MPH-1, Sim-2 and the like were discovered.
PTDs are known to perform the efficient delivery of molecules in vitro or in vivo regardless of the kind thereof or the cell type. Most PTDs can form a stable non-covalent bond with nucleic acids and deliver the nucleic acids into cells. The third helix of Antennapedia homeodomain has been shown to form stable non-covalent complexes with small oligonucleotides and to facilitate their internalization (Dom, G., et al., Nucleic Acids Res. 31:556-561 (2003)). Pep-3 has been reported to form stable complexes with peptide nucleic acid through non-covalent interactions, and promote their delivery into cells (Morris, M. C., et al., Nucleic Acids Res. 35 (2007)). MPG peptide, which contains a hydrophobic domain derived from the fusion sequence of HIV gp41 and a hydrophilic domain derived from the nuclear localization sequence of SV40 T-antigen, has been demonstrated to form non-covalent bond with antisense oligonucleotides to deliver the oligonucleotides into cultured mammalian cells (Morris, M. C. et al., Nucleic Acids Res. 25:2730-2736 (1997)). A dimer, trimer and tetramer of Tat peptide have been reported to form stable particles with plasmid DNA through non-covalent interactions, and promote their delivery into cells (Rudolph, C., et al., J. Biol. Chem. 278:11411-11418 (2003)).
PTDs can also form a stable covalent bond with nucleic acids to promote their delivery. Tat peptide covalently attached to liposomes promotes rapid delivery of DNA (Torchilin, V. P., et al., Proc. Natl. Acad. Sci. USA 98:8786-8791 (2001), and Torchilin, V. P., et al., Proc. Natl. Acad. Sci. USA 100:1972-1977 (2003)). The use of penetratin and transportan to deliver peptide nucleic acid molecules across plasma membranes, through a labile bond, such as a disulfide bond, has also been described (see U.S. Pat. No. 6,025,140).
A PTD forming a covalent bond with siRNA has been described. Tat peptide covalently attached to siRNA promotes nuclear delivery of siRNA (Chiu, Y., et al., Chem. Biol. 11(8):1165-1175 (2004)). Penetratin and transportan peptide covalently attached to siRNA promote efficient cellular delivery of siRNA (Davidson, T. J., et al., J. Neurosci. 24(45):10040-10046 (2004); Muratovska, A. and Eccles, M. R., FEBS 558:63-68 (2004)).
Although high efficiency in delivery is achieved in some studies, the use of PDT in delivery of siRNA or DNA vectors for siRNA does not produce RNAi effects of stable and high efficiency. Also, there are reports that the use of PDT provides insignificant effects. siRNA covalently attached to Tat or penetratin peptide showed no RNAi effects in vivo (Moschos, S. A., et al., poster presentation at the Biochemical Society Focused Meeting, UK, (2007)).
As mentioned above, most PTDs are rich in positively charged amino acids, which can bind to the negatively charged phosphate backbone of nucleic acids. This binding between nucleic acids and PTDs is possible both when PTDs and nucleic acids form a non-covalent bond and when they form a covalent bond. Most studies employed only a single PTD unit. Even weak binding between the PTD and the nucleic acid can influence the structure of PTD which is important to maintain its delivery function. Thus, the delivery efficiency of a PTD can be greatly reduced. A PTD, which does not bind to nucleic acids, maintains the ability to be delivered into cells, but cannot be used to deliver nucleic acids. Also, a PTD that binds to nucleic acids, has a reduced ability to be delivered into cells. As a result, methods of delivering nucleic acids into cells using PTDs in prior studies are not effective or efficient.
Accordingly, the present inventors designed multimeric or spacer-incorporated PTD molecules to deliver nucleic acids into cells, whereby the multimeric or spacer-incorporated PTD molecules maintain the ability to be delivered into cells.